Neurotox and Cardiac Safety Assessment: Case

Studies Employing iPS Cell lines and Next Generation

MEA Technology

Workshop Outline

In vitro Assessments of Drug-induced Neuronal Modulationand MEA-based Seizure Prediction

Blake Anson, PhD Cellular Dynamics International

Comprehensive In-vitro Proarrhythmia Assay (CiPA)MEA and hiPSC-cardiomyocytes as reproducible and predictive tools for detecting proarrhythmia

Hong Shi, PhD Bristol-Myers Squibb

Get Ready to LEAPTM

Mike Clements, PhD AXION Biosystems

Please use this link to access the seizure prediction slides

Please use this link to access more information on iCell Cardiomyocytes and Cardiomyocytes2

Please use this link to learn more about the Maestro Edge

In vitro Assessments of Drug-induced Neuronal Modulationand MEA-based Seizure Prediction

Blake Anson, PhD Cellular Dynamics International

Please use this link to access the slides

Comprehensive In-vitro Proarrhythmia Assay (CiPA)

MEA and hiPSC-cardiomyocytes as reproducible and predictive tools for detecting proarrhythmia

Hong Shi, MD BMS

Talk Outline

Comprehensive In-vitro Proarrhythmia Assay (CiPA)

System stability and cross site reproducibility

• CiPA Paradigm

• MEA Tracings and Drug Effects

• Baseline and Control Responses• Data from BMS

• CiPA Compound Responses• Data from BMS

• Multi-site Correlations

• Summary

iCell Cardiomyocytes2

MaestroMultiwell MEA Platform

Short CiPA Overview

Comprehensive In-vitro Proarrhythmia Assay (CiPA)

Cross site correlations

Human iPSC-derived cardiomyocytes and MEA recordings detect

electrophysiological phenotypes related to key mechanistic effects.

MEA Tracings

Phenotypes of hiPSC-CM Electrophysiology

• Baseline histograms were compiled across 14 separate 48-well plates for BP, AMP, FPDc (Fridericia), and BP Coefficient of Variability.

• BP CoV and AMP passed the CiPAPhase 2 Protocol inclusion criteria.

• BP, BP CoV, and FPDc were highly reliable across plates for the study.

Human iPSC-derived cardiomyocytes and MEA recordings provide a stable

platform for detect electrophysiological phenotypes

Baseline Data

Safety assays require a consistent phenotype across wells and plates

The label-free assay supports reliable and minimal responses to vehicle

control responses, enabling sensitive detection of the positive control.

0.5nM Dofetilide- IC50 ~ 12nM1

- Used a submaximal

concentrations for

maximal system

sensitivity1Snyders and Chaudhary,

1996

Negative control- Inter-plate stability

- Also used as experimental check

Vehicle and Positive Controls

Detection of positive control compounds defines assay sensitivity

Compound selection was determined by experts and designed to cross

multiple classifications with concentrations that bracket the therapeutic level

CiPA Phase 2 Compounds

Detection of positive control compounds defines assay sensitivity

Low risk compounds generally had little effect on FPDc or elicited a shortening of FPDc.

Intermediate risk compounds ranged from minimal to significant prolongation of FPDc.

High risk compounds consistently induced significant prolongation.

Human iPSC-derived cardiomyocytes and MEA recordings show appropriate

compound classification for arrhythmogenic potential

Low Risk Intermediate Risk High Risk

0/9 compounds showed FPDc

prolongation >50% by 30x

Cmax

Note: EADs included for FPDc calculation

5/11 compounds

showed FPDc

prolongation

>50% by 30x

Cmax

7/8 compounds showed

FPDc prolongation >50%

by 30x Cmax

Compound ResponsesBMS Data

Data from BMS matched well with that from multiple sites (Eisai, Genentech, Axion)

Overall the test system shows good multi-site reproducibility

Data divergence generally occurred at >30X Cmax, or upon incidence of EADs

Low Risk Intermediate Risk High Risk

Human iPSC-derived cardiomyocytes and MEA recordings show reproducible

classification results across multiple sites

Compound ResponsesMulti-Site Data

• Each point in the scatter plot represents the percent change in FPDc at two sites for a single compound and concentration.

• The data show extremely high correlation for conditions that do not elicit EADs, and good correlation even when EADs are present.

• The CiPA Phase 2 data was highly correlated across all sites using the CDI/Maestro cell-platform combination.

BMS AXN GNE ESI

BMS 1 0.94 0.95 0.94

AXN 0.94 1 0.89 0.92

GNE 0.95 0.89 1 0.95

ESI 0.94 0.92 0.95 1

Blinova et al. submitted

Human iPSC-derived cardiomyocytes and MEA recordings show reproducible

quantitative data across multiple sites

Multi-Site Correlations

Site-to-site consistency highlights a reliable assay

Risk classification of Ando et al, 2016- TdP risk (y-axis) based on FPDc- Ratio (x-axis) based on concentration ratio of in-vitro effect/ clinical level

Red = high risk

Yellow = intermediateGreen = low risk

Ando et al., 2016 J Pharm Tox Meth

AXION FPDc

Sensitivity = 0.79Specificity = 0.78Accuracy = 0.79

Sensitivity = 0.84Specificity = 0.67Accuracy = 0.79

BMS FPDc

Human iPSC-derived cardiomyocytes and MEA datasets will enable the

generation and refinement of in-vitro tools to predict proarrhythmia

•Current data set is consistent with previous classification resultsSmaller data set, unequal grouping

• Additional assessment paradigms will continue to emerge

Data Interpretation

Current results can be mapped onto existing scheme(s)

• Positive Controls

• Reproducible (see left)

• Were used at sub-maximal concentrations

(0.5nM Dofetilide) to demonstrate

detection of sensitive effects

• Compound Responses

• Highly correlated across sites (≥ 0.89)

• Risk Classification was robust across sites

• Translated well to available clinical data

• Baseline data was reproducible• Across MEA plates

• Experimental days

• Experimental sites

• Vehicle controls• Were highly reproducible (see above)

• Acted as an internal system check

Human iPSC-derived cardiomyocytes and MEA recordings provide an

efficient, robust, and translatable in-vitro paradigm for predicting

proarrhythmia

Summary

Site-to-site consistency highlights a reliable assay

03.12.18 Society of Toxicology

Field Potential Signal for the CM-MEA Assay

Relationship to the Cardiomyocyte Signal Spectrum

Clinical ECG

Action Potential

Field Potential

LEAP

Field Potential

Field

Potential

LEAP

Local Extracellular Action Potential (LEAP)

How does it work?

Adapted from Borkholder, 1998

LEAP

Field Potential

Sealing Resistance GΩ

Re

co

rde

d A

mp

litu

de

(m

V)

Transmembrane Potential

Patent Pending

Maestro Pro & Edge

World’s most advanced MEA platforms

Maestro Pro & EdgeBioCore v4

Local Extracellular Action Potential (LEAP)

Signal Specifications

5-20 mV

Peak-to-PeakSNR ~1000+

Stable for 10-20+ minutes after induction

10 mV

FP at Same Scale

The LEAP Advantage #1

FP to AP “Translation”

500uV5mV

5mV

The LEAP signal provides a direct mapping from field

potential to action potential morphology

FP and LEAP Signals from the Same Wells, 10x Zoom on the FP

Depolarization Repolarization EADs

The LEAP Advantage #2

Arrhythmia/EAD Detection

The LEAP signal improves the accuracy of automated

analysis, and allows automation of EAD detection

The LEAP Advantage #2

Arrhythmia/EAD Detection

The updated CiPA Analysis Tool provides automated EAD

detection for LEAP signals, as well as other LEAP endpoints.

The LEAP Advantage #3

LEAP Morphology

The LEAP signal provides additional and complementary

metrics to the standard CM-MEA field potential assay

LEAP Duration (LEAPD)

5mV

The induction of LEAP does not affect the underlying

electrophysiologic properties of the cardiomyocyte syncytium.

The LEAP Advantage #4

LEAP does not disrupt the underlying biology

Implementing the LEAP Assay

2mV100uV

800m

s

Baseline Dosed LEAP

LEAP Case Study on Selective Blockers

L-Type Calcium Block

(Nifedipine)

hERG Potassium Block

(E-4031)

Multi-Ion Channel Block

(Verapamil)

LEAP Case Study on Selective Blockers

LEAP Case Study on Tolterodine vs. Terodiline

To

ltero

din

eTe

rod

ilin

e

Martin et al, 2006

“Next Steps” Combining LEAP with Other Maestro Technology

CytoView MEA 24-well

Electrical Pacing

Maestro Pro Enables Next Generation Applications

• The Local Extracellular Action Potential (LEAP) signal adds a new dimension to the

standard CM-MEA assay

• A simple, label-free induction phase produces the LEAP signal, which is characterized

by:

• An action potential-like waveform with high amplitude

• Stability on the time scale of 10-20+ minutes

• LEAP enables:

• Clear translation of field potential signals to action potential signals

• Improvements in automated EAD detection

• Analysis of LEAP morphology to provide additional end points

• LEAP does not disrupt the underlying biology

Announcing the LEAP Assay for use in Spring 2018!

Acknowledgments

Daniel Millard

Heather Hayes

Anthony Nicolini

Colin Arrowood

Jim Ross

Visit www.axionbio.com

For more information on

the

Maestro ProTM and EdgeTM

Booth #1342Tuesday March 13 | 1:30 - 3:30pm

Poster P691

Learn more about LEAP